Glucoamylase (1,4-.alpha.-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme which catalyzes the release of D-glucose from the nonreducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts, with those from Aspergilli being commercially most important.
Commercially, the glucoamylase enzyme is used to convert cornstarch which is already partially hydrolyzed by .alpha.-amylase to glucose. The glucose is further converted by glucose isomerase to a mixture composed almost equally of glucose and fructose. This mixture, or the mixture further enriched with fructose, is the commonly used high fructose corn syrup commercialized throughout the world. This syrup is the world's largest tonnage product produced by an enzymatic process. The three enzymes involved in the conversion of starch to fructose are among the most important industrial enzymes produced, even though two of them, .alpha.-amylase and glucoamylase, are relatively inexpensive on a weight or activity basis.
Two main problems exist with regard to the commercial use of glucoamylase in the production of high fructose corn syrup. The first problem is with regard to the thermal stability of glucoamylase. Glucoamylase is not as thermally stable as .alpha.-amylase or glucose isomerase and it is most active and stable at lower pH's than either .alpha.-amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH. Second, at the high solids concentrations used commercially for high fructose corn syrup production, glucoamylase synthesizes di-,tri-, and tetrasaccharides from the glucose that is produced. Accordingly, the glucose yield does not exceed 95% of theoretical. By quantity, the chief by-product formed is isomaltose, a disaccharide containing two glucosyl residues linked by an .alpha.-(1.fwdarw.6) bond. A glucoamylase that can produce glucose without by-products would be of great commercial potential if its cost were not significantly higher than that of the current enzyme being produced, which is made by the two very closely related fungal species Aspergillus niger and Aspergillus awamori. The glucoamylases from these two sources are identical.
Glucoamylases from a variety of fungal sources have been sequenced and have high homology (1,2). The high homology between the variety of fungal sources suggests that the enzymes are all structurally and functionally similar. Furthermore, kinetic measurements on a number of glucoamylases have demonstrated that their subsite binding energies are almost identical (3,4,5,6,7).
Applicant has conducted studies of the homology of amino acids from identical A. niger and A. awamori glucoamylases, both with other glucoamylases and with other enzymes that hydrolyze starch and related substances (8). This was done to identify amino acids that were common to enzymes that cannot cleave .alpha.-(1.fwdarw.6) glucosidic bonds (chiefly .alpha.-amylases) from those that can hydrolyze .alpha.-(1.fwdarw.6) glucosidic bonds (glucoamylases and isomaltase).
Applicant has found that glucoamylase is represented in three out of six regions of sequence similarity among several starch hydrolases (8). It has been determined that Region 1 from A. niger glucoamylase residues 109-122, Region 4 from glucoamylase residues 172-184, and Region 6 from residues 382-398 contain these sequence similarities. The regions represent sequence similarities among enzymes cleaving only .alpha.-(1.fwdarw.4) bonds, enzymes cleaving only .alpha.-(1.fwdarw.6) bonds, and glucoamylase, which cleaves both. Amino acids at positions 178, 182, 183 and 184 differed between the groups, which suggested changing amino acids at these positions. Applicant has also noted homology at position 119. By utilizing cassette mutagenesis, applicant made substitutions of amino acids at these various positions consistent with the homology studies (8).
In connection with the fourteenth ICS meeting in Stockholm in 1988, applicant presented a poster disclosing that site-directed mutagenesis supports the participation of Tyr116 and Trp120 in substrate binding and Glu180 in catalysis. Moreover, a role was suggested for Trp170 in isomaltose binding, but this aspect remains to be studied by site- directed mutagenesis. The poster also disclosed that the mutation of Asn182 to Ala provided an active enzyme, but no results were disclosed or suggested regarding relative specificity of that enzyme.
As stated above, a drawback in the industrial use of glucoamylase is that D-glucose yields are limited to approximately 95% in concentrated starch solutions. This occurs because of the slow hydrolysis of .alpha.-(1.fwdarw.6)-D-glucosidic bonds in starch and the formation of various accumulating condensation products, mainly .alpha.-(1.fwdarw.6)-linked isomaltooligosaccharides, in a stepwise manner from D-glucose (9). A reduction of the rate that glucoamylase cleaves and therefore forms .alpha.-(1.fwdarw.6) bonds relative to the rate it cleaves .alpha.-(1.fwdarw.4) bonds has practical implications. Mutations at Trp120, Asp176, Glu179 and Glu180 in A. awamori glucoamylase all were critical for enzyme activity (10, 11). Applicant proceeded to investigate further amino acid mutations in order to increase the selectivity of glucoamylase for maltose over isomaltose hydrolysis. These experiments are problematic since the three-dimensional structure of glucoamylase has not been determined. Instead, primary use was made of regional sequence similarities with glucoamylases other than those produced by A. awamori and A. niger as well as with other enzymes active on .alpha.-(1.fwdarw.4)- and .alpha.-(1.fwdarw.6)-linked D-glucosyl oligo- and polysaccharides (FIG. 1).
Applicant thus conducted tests, for example involving mutations of Ser119, Leu177, Trp178, Asn182, Gly183, and Ser184.
In Region 1 (FIG. 1) , the glucoamylases at positions corresponding to A. niger 119 have either Ser, Ala or Pro where the .alpha.-amylases and cyclodextrin glucanotransferases (CGTase) all have Tyr. Therefore, Ser119 of A. niger glucoamylase was mutated to Tyr so it would resemble the .alpha.-amylases and CGTases.
In Region 4, Leu177 was mutated to His, since enzymes active on .alpha.-(1.fwdarw.6)glucosidic bonds characteristically contain amino acid residues with smaller aliphatic side chains at this homologous position, while enzymes active only at .alpha.-(1.fwdarw.4)-D-glucosidic bonds contain primarily Phe or Trp, which have large aromatic side chains. Ile, Val and Leu also occur at this position.
At residue 178 in A. niger glucoamylase Trp was mutated to Arg because Trp was conserved in the glucoamylases and isomaltase which cleave .alpha.-(1.fwdarw.6) bonds, but Arg is found in all of the .alpha.-amylases, maltases, CGTase, amylomaltase and branching enzyme which do not.
Asn182 was mutated to Ala based on similar comparisons because Asn was conserved in all of the glucoamylases and isomaltase but was replaced with residues containing short aliphatic side chains such as Ala, Val, and Ser, usually Ala, in most of the .alpha. amylases.
At A. niger glucoamylase position 183, the glucoamylases all have Gly, isomaltose has an acidic side chain Glu, while the enzymes cleaving only .alpha.-(1.fwdarw.4) glucosidic bonds have a basic side chain, primarily Lys, although Arg also occurs. Branching enzyme is the sole .alpha.-(1.fwdarw.4) acting enzyme which does not have a basic group at this position, but instead has Ala there.(8) Therefore, Gly183 was changed to Lys.
At position 184, the glucoamylases have Ser, Val and Met, while isomaltase also has Val. However, the enzymes cleaving .alpha.-(1.fwdarw.4) bonds contain predominantly His at this position, though Gly, Leu, Gln, and Ser also occur. Therefore, Ser184 was changed to His.
Pursuant to the present invention, all of the above changes were constructed to make glucoamylase resemble more closely the .alpha.-(1.fwdarw.4) bond hydrolyzing enzymes and less closely the .alpha.-(1.fwdarw.6) bond hydrolyzing enzymes.
The Leu177.fwdarw.His mutation resulted in a loss of selectivity and the Trp178.fwdarw.Arg mutation resulted in increased selectivity, but with substantial losses in activity, whereas applicant found increased relative selectivity of hydrolysis of .alpha.-(1.fwdarw.4) bonds over .alpha.-(1.fwdarw.6) bonds with either only a small loss of activity or none at all with mutations at the 119, 182, 183, and 184 positions. These results provide highly significant commercial potential.